Peptide showing melanogenesis promoting activity and use thereof

ABSTRACT

The present invention provides a peptide exhibiting melanogenesis promoting activity. A peptide according to the present invention increases the activity and expression of tyrosinase and the expression of factors involved in melanogenesis, thereby exhibiting an outstanding effect on melanogenesis. The peptide of the present invention can be used for the prevention, alleviation, and treatment of hypomelanosis. The outstanding activity and stability stated above allow the peptide of the present invention to be very favorably applied to medicines, quasi-medicines, and cosmetics.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Nov. 24, 2020 isnamed 51401-015002_Sequence_Listing_11.24.20_ST25 and is 2,114 bytes insize.

TECHNICAL FIELD

The present invention relates to a peptide exhibiting melanogenesispromoting activity; a pharmaceutical composition containing the peptideas an active ingredient, for prevention and/or treatment ofhypomelanosis; a cosmetic composition containing the peptide as anactive ingredient, for prevention and/or alleviation of hypomelanosis;and a use of the peptide for prevention, alleviation, and/or treatmentof hypomelanosis.

BACKGROUND ART

In skin cells, melanin is produced in melanosomes of melanocytes in theepidermal stratum basale as a defense mechanism against the stimuli ofultraviolet (UV) light, environmental pollution, and other externalfactors. Melanin is an important factor in determining the color of theskin, eyes, and hair of animals. Hypomelanosis is also known as a riskfactor for skin cancer. East Asians are sensitive to the overproductionof melanin, and thus much research has been conducted on whitening inwhich melanogenesis is inhibited. In recent years, the demand fortreatment of vitiligo, which is caused by melanogenesis inhibition, isalso increasing, and thus research thereon is in progress.

Vitiligo is an acquired depigmentation disease in which milky spots ofvarious sizes and shapes appear due to apoptosis or necrosis ofmelanocytes. Vitiligo is a relatively common disease that occurs inabout 1% of the world's population, and there is no difference in thedisease according to race or region. Regarding the age of occurrence,vitiligo occurs most frequently at ages of 10 to 30 years, with 95% ofcases occurring before the age of 40, and 30% of patients having afamily history.

The cause of vitiligo has not yet been accurately identified, but thereare various theories, such as the autoimmune theory, the neural theory,and melanocyte self-destruction theory. The autoimmune theory is thatthe destruction or dysfunction of melanocytes is caused by theexpression of auto-antibodies to melanocyte-based antigens, ormelanocytes are destroyed by lymphokines secreted by cytotoxiclymphocytes or activated lymphocytes. The neural theory is that hydrogenperoxide associated with stress is generated due to abnormalcatecholamine biosynthesis and increased monoamine oxidase, resulting inthe destruction of melanocytes, and vitiligo may occur along theganglion or vitiligo may occur after nerve damage or stress. Themelanocyte self-destruction theory is that intermediate metabolites orphenol complexes as final metabolites of the melanogenic processaccumulate in melanocytes, resulting in cell destruction. In addition,various factors, such as inherent cellular defects, genetic factors,apoptosis, calcium metabolism disorders, have been suggested.

Melanin is synthesized in melanocytes, and plays an important role inskin protection against UV irradiation or the absorption of toxicsubstances and chemical substances. Therefore, in people whose melaninsynthesis does not occur normally, there is a problem with appearance inthat part of the skin, rather than all of the skin, becomes white,causing blotches, and there is a severe problem of being sensitive toexternal stimuli.

Tyrosinase, tyrosinase related protein-1 (TRP-1), and tyrosinase relatedprotein-2 (TRP-2), which are important enzymes in melanin synthesis, actas catalysts for oxidative reactions (Pigment Cell Res. 14 (6): 43744).

Here, tyrosinase acts to oxidize tyrosine intoL-3,4-dihydroxyphenylalanine (DOPA) and DOPA into DOPA quinone, andTRP-1 is a dihydroxyindole carboxylic acid oxidase that is involved inthe conversion of 5,6-dihydroxyindole-2-carboxylic acid (DHICA) intoindol-5,6-quinone-2-carboxylic acid. TRP-1 also serves to stabilizetyrosinase and regulate activity thereof. TRP-2, which is a DOPA chrometautomerase, converts DOPA chrome into DHICA to form eumelanin andpheomelanin, which constitute melanocytes, and the ratio thereofdetermines the colors of skin, hair, and eyes.

Melanin synthesis is activated by UV irradiation and α-melanocytestimulating hormone (MSH). Here, α-MSH, which is a peptide hormone, isknown to be produced by UV light and made from several cells includingthose of the pituitary gland and skin.

Here, α-MSH acts on melanocortin receptors (MCR) of melanocytes byparacrine signaling to regulate the activity of the transcriptionfactor, microphthalmia-associated transcription factor (MITF), therebyregulating the activity of tyrosinase, DHICA oxidase (TRP-1), andDOPAchrome tautomerase (TRP-2), which play important roles in melaninsynthesis (THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 273, No. 31, Issueof July 31, pp. 195609565, 1998).

It has been reported that the stimulation of melanocytes by UV or α-MSHleads to the activation of tyrosinase by p38 or protein kinase A (PKA),respectively. In these two pathways, especially, the α-MSH→cAMP→PKApathway plays an important role in melanin synthesis. The increase incAMP stimulates the phosphorylation of cAMP-responsive element bindingprotein (CREB), increasing the expression of the transcription factorMITF, which enhances the activity of tyrosinase and increases the mRNAexpression of tyrosinase (Nucleic Acids Res. 30 (14): 3096106, PigmentCell Melanoma Res 21 (6): 66576).

Many East Asian people including Koreans want to have light skin color,and thus much research has been conducted on whitening components whichinhibit melanogenesis. However, melanin is synthesized in melanocytes inthe skin, and plays an important role in skin protection from UVirradiation or the absorption of toxic substances and chemicalsubstances. Since the absence of normal synthesis of melanin makes theskin sensitive to external stimuli and results in an abnormal externalappearance, there is a need to treat melanin synthesis so that melaninsynthesis can be normalized, and studies on this have also beenconducted. However, so far, the development of technology for promotingmelanin synthesis has not been carried out sufficiently.

DESCRIPTION OF EMBODIMENTS Technical Problem

The present inventors endeavored to develop peptides capable ofpromoting melanogenesis, and as a result, the present inventorsconfirmed that a peptide including a sequence selected from the groupconsisting of the acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQID NO: 3 has excellent melanogenesis promoting activity and establishedthat these peptides may be useful in the prevention and treatment ofhypomelanosis, and thus the present inventors completed the presentinvention.

Therefore, provided is a peptide showing melanogenesis promotingactivity, the peptide including the amino acid sequence of SEQ ID NO: 1,SEQ ID NO: 2, or SEQ ID NO: 3.

Provided is a pharmaceutical composition for preventing and/or treatinghypomelanosis, the pharmaceutical composition containing, as an activeingredient, at least one peptide selected from the group consisting ofpeptides each including a sequence selected from the group consisting ofthe amino acid sequence of SEQ ID NO: 1, the amino acid sequence of SEQID NO: 2, and the amino acid sequence of SEQ ID NO: 3.

Provided is a cosmetic composition for prevention and/or alleviation ofhypomelanosis, the cosmetic composition containing, as an activeingredient, at least one peptide selected from the group consisting ofpeptides each including a sequence selected from the group consisting ofthe amino acid sequence of SEQ ID NO: 1, the amino acid sequence of SEQID NO: 2, and the amino acid sequence of SEQ ID NO: 3.

Solution to Problem

According to an aspect of the present invention, the present inventorshave tried to develop a peptide capable of promoting melanogenesis, andas a result, it was confirmed that a peptide containing the amino acidsequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 has excellentmelanogenesis promoting activity and established that the peptide may befavorably used in the prevention and treatment of hypomelanosis.

Thus, the present invention is related to a peptide showingmelanogenesis promoting activity; a pharmaceutical compositioncontaining the peptide as an active ingredient for prevention and/ortreatment of hypomelanosis; a cosmetic composition containing thepeptide as an active ingredient for prevention and/or alleviation ofhypomelanosis; and a use of the peptide for prevention, alleviation,and/or treatment of hypomelanosis.

Hereinafter, the present invention will be described in further detail.

According to an embodiment of the present invention, a peptide includesthe amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

The peptide according to an embodiment may include the amino acidsequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, and, forexample, may consist of the amino acid sequence of SEQ ID NO: 1, SEQ IDNO: 2, or SEQ ID NO: 3.

The peptide may show melanogenesis promoting activity.

The peptide of the present invention is obtained by screening peptides,which have excellent melanogenesis promotion effects, from peptidelibraries possessed by the present inventors, through experiments aboutgene and protein expression changes, and a total of three types ofpeptides are provided as a peptide of the present invention.

As used herein, the term “peptide” refers to a linear molecule formed ofamino acid residues link to each other via peptide bonds. The peptide ofthe present invention may be prepared by known chemical synthesismethods, especially, solid-phase synthesis techniques (Merrifield, J.Amer. Chem. Soc. 85:2149-54(1963); Stewart, et al., Solid Phase PeptideSynthesis, 2nd. ed., Pierce Chem. Co.: Rockford, 111 (1984)) orliquid-phase synthesis techniques (U.S. Pat. No. 5,516,891).

The peptide of the present invention may have a modification induced atthe N-terminal and/or C-terminal thereof in order to select a part of anamino acid sequence and increase the activity thereof.

The N-terminal and/or C-terminal modification of the peptide improvesthe stability of the peptide, and this modification allows the peptideof the present invention to have an increased half-life at the time ofin vivo administration, thereby having a high half-life.

Also, the C-terminal and/or N-terminal modification may protect thepeptide of the present invention from in vivo protein cleavage enzymesor may increase binding force to a receptor.

For example, the C-terminal modification may be a modification of theC-terminal of the peptide into a hydroxyl group (—OH), an amino group(—NH₂), or an azide group (—NHNH₂), but embodiments are not limitedthereto.

The N-terminal modification may be an attachment of at least oneprotecting group selected from the group consisting of an acetyl group,a fluorenyl methoxy carbonyl group, a formyl group, a palmitoyl group, amyristyl group, a stearyl group, and polyethylene glycol (PEG) to theN-terminal of the peptide, but embodiments are not limited thereto.

As used herein, the term “stability” refers to storage stability (e.g.,room-temperature stability) as well as in vivo stability.

According to an aspect of the present invention, the peptide of thepresent invention increases the melanogenesis in melanocytes, increasesthe activity and expression of tyrosinase, which is an enzyme forregulating melanin synthesis, increases the expression of MITF and TRP1and increases the phosphorylation of CREB, these being factors involvedin melanogenesis.

These results indicate that the peptide of the present invention has aneffect of relieving vitiligo by increasing melanogenesis. Therefore, thepeptide of the present invention can be used for the prevention,alleviation, and/or treatment of hypomelanosis.

In the present invention, the hypomelanosis may be vitiligo, albinism,nevus depigmentosus, pityriasis alba, pityriasis versicolor,post-inflammatory depigmentation, morphea, piebaldism, idiopathicguttate hypomelanosis, or leucoderma punctatum.

According to another aspect of the present invention, a pharmaceuticalcomposition for prevention or treatment of hypomelanosis contains, as anactive ingredient, at least one peptide selected from the groupconsisting of a peptide consisting of the amino acid sequence of SEQ IDNO: 1, a peptide consisting of the amino acid sequence of SEQ ID NO: 2,and a peptide consisting of the amino acid sequence of SEQ ID NO: 3.

Since the pharmaceutical composition of the present invention containsthe foregoing peptide of the present invention as an active ingredient,the descriptions of overlapping contents therebetween will be omitted toavoid excessive complexity of the present specification.

The pharmaceutical composition of the present invention may contain apharmaceutically effective amount of the foregoing peptide of thepresent invention.

As used herein, the term “pharmaceutically effective amount” refers toan amount sufficient to attain the efficacy or activity of the foregoingpeptide.

The pharmaceutical composition may further include a pharmaceuticallyacceptable carrier, but embodiments are not limited thereto.

Examples of the pharmaceutically acceptable carrier include lactose,dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calciumphosphate, alginate, gelatin, calcium silicate, microcrystallinecellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc,magnesium stearate, and/or mineral oil, but embodiments are not limitedthereto.

The pharmaceutical composition of the present invention may furtherinclude a lubricant, a wetting agent, a sweetening agent, a flavoringagent, an emulsifier, a suspending agent, and a preservative, inaddition to the above ingredients.

The pharmaceutical composition of the present invention may be in theform appropriate for the desired administration method.

In terms of the pharmaceutical composition of the present invention, theexpression “administration” denotes introducing a predetermined materialto a patient by using an appropriate method.

The pharmaceutical composition of the present invention may beadministered through a general rout as long as the drug may reach thetarget tissue. For example, the drug may be administered byintraperitoneal administration, intravenous administration,intramuscular administration, subcutaneous administration, intradermaladministration, oral administration, topical administration, intranasaladministration, pulmonary administration, or rectal administration. Insome embodiments, the drug may be administered, preferably, byparenteral administration or, more preferably, by topical skinadministration.

Also, the pharmaceutical composition of the present invention may beadministered by using a device that may transfer the active ingredientto a target cell.

The appropriate dose of the pharmaceutical composition of the presentinvention varies depending on factors, such as a formulating method, amanner of administration, patient's age, body weight, gender, morbidity,food, a time of administration, a route of administration, an excretionrate, and response sensitivity. The ordinarily skilled practitioners mayeasily determine and prescribe the dose that is effective for thedesired treatment or prevention. According to a preferable embodiment ofthe present invention, the daily dose of the pharmaceutical compositionof the present invention is 0.001 to 1000 mg/kg.

The pharmaceutical composition of the present invention is formulatedusing a pharmaceutically acceptable carrier and/or excipient accordingto a method that is easily conducted by a person having ordinary skillsin the art to which the present invention pertains, and thepharmaceutical composition of the present invention may be prepared intoa unit dosage form or may be inserted into a multi-dose container.

Here, the dosage form may be a solution in an oily or aqueous medium, asuspension, an emulsion, an extract, a powder, granules, a tablet, acapsule, or a gel (e.g., a hydrogel), and may further contain adispersing agent or a stabilizer.

According to another aspect of the present invention, a cosmeticcomposition for preventing or alleviating hypomelanosis contains, as anactive ingredient, at least one peptide selected from the groupconsisting of a peptide consisting of the amino acid sequence of SEQ IDNO: 1, a peptide consisting of the amino acid sequence of SEQ ID NO: 2,and a peptide consisting of the amino acid sequence of SEQ ID NO: 3.

The cosmetic composition of the present invention may include acosmetically effective amount of the foregoing peptide of the presentinvention.

As used herein, the term “cosmetically effective amount” refers to anamount sufficient to attain the efficacy of the foregoing composition ofthe present invention.

In addition, the cosmetic composition may further include a cosmeticallyacceptable carrier, but embodiments are not limited thereto.

The cosmetic composition of the present invention may be formulated intoany form that is commonly prepared into, and examples of the form mayinclude a solution, a suspension, an emulsion, a paste, a gel, a cream,a lotion, a powder, a soap, a surfactant-containing cleanser, an oil, apowder foundation, an emulsion foundation, a wax foundation, and/or aspray, but embodiments are not limited thereto. More specifically, thecosmetic composition of the present invention may be prepared in theform of emollient lotion, nourishing lotion, nourishing cream, massagecream, essence, eye cream, cleansing cream, cleansing foam, cleansingwater, pack, spray, and/or powder.

In cases where the form of the cosmetic composition is a paste, cream,or gel, useful examples of the carrier ingredient may include an animaloil, a plant oil, wax, paraffin, starch, tragacanth, a cellulosederivative, polyethylene glycol, silicone, bentonite, silica, talc,and/or zinc oxide, but embodiments are not limited thereto.

When the form of the cosmetic composition is a powder or spray, lactose,talc, silica, aluminum hydroxide, calcium silicate, or a polyamidepowder may be used as a carrier ingredient, but embodiments are notlimited thereto. Especially, when the form of the cosmetic compositionof the present invention is a spray, the spray may further include apropellant, such as chlorofluorohydrocarbon, propane/butane, and/ordimethyl ether, but embodiments are not limited thereto.

When the form of the cosmetic composition is a solution or emulsion, asolvent, solubilizer, or emulsifier may be used as a carrier component,and examples thereof include water, ethanol, isopropanol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butyl glycol oil, glycerol aliphatic esters, polyethyleneglycol, and/or fatty acid esters of sorbitan.

When the form of the cosmetic composition is a suspension, examples ofthe carrier ingredient may include a liquid diluent (such as water,ethanol, and/or propylene glycol), a suspending agent (such asethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, and/orpolyoxyethylene sorbitan ester), microcrystalline cellulose, aluminummetahydroxide, bentonite, agar, and/or tragacanth, but embodiments arenot limited thereto.

When the form of the cosmetic composition is a surfactant-containingcleanser, examples of the carrier ingredient may be aliphatic alcoholsulfate, aliphatic alcohol ether sulfate, sulfosuccinate monoester,isethionate, imidazolium derivatives, methyl taurate, sarcosinate, fattyacid amide ether sulfate, alkyl amido betaine, aliphatic alcohol, fattyacid glyceride, fatty acid diethanolamide, plant oil, lanolinederivatives, and/or ethoxylated glycerol fatty acid ester, butembodiments are not limited thereto.

In addition to the peptide and carrier ingredients, components containedin the cosmetic composition of the present invention as the activeingredients may include ingredients ordinarily used in cosmeticcompositions, and examples thereof may include ordinary supplements,such as an antioxidant, a stabilizer, a solubilizer, vitamins, apigment, and/or a flavoring agent, but embodiments are not limitedthereto.

Advantageous Effects of Disclosure

The present invention is directed to a peptide showing melanogenesispromoting activity; a pharmaceutical composition including the peptideas an active ingredient for prevention and/or treatment ofhypomelanosis; a cosmetic composition including the peptide as an activeingredient for prevention and/or alleviation of hypomelanosis; and a useof the peptide for prevention, alleviation, and/or treatment ofhypomelanosis. The peptide of the present invention increases theactivity and expression of tyrosinase and increases the expression offactors involved in melanogenesis, thereby exhibiting excellent effectsin melanogenesis, and may be very favorably applied to medicines,quasi-medicines, and cosmetics through excellent activity and safetythereof.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a graph that confirms a melanogenesis increasing effect of apeptide consisting of the amino acid sequence of SEQ ID NO: 1 accordingto an embodiment of the present invention;

FIG. 2 is a graph that confirms a tyrosinase increasing effect of apeptide consisting of the amino acid sequence of SEQ ID NO: 1 accordingto an embodiment of the present invention;

FIG. 3 shows a melanogenesis-related gene expression increasing effectof a peptide consisting of the amino acid sequence of SEQ ID NO: 1according to an embodiment of the present invention;

FIG. 4 shows a melanogenesis-related protein expression increasingeffect of a peptide consisting of the amino acid sequence of SEQ ID NO:1 according to an embodiment of the present invention;

FIG. 5 shows an effect of increased phosphorylation of cAMP-responsiveelement binding protein (CREB) of a peptide consisting of the amino acidsequence of SEQ ID NO: 1 according to an embodiment of the presentinvention;

FIG. 6 is a graph that confirms a melanogenesis increasing effect of apeptide consisting of the amino acid sequence of SEQ ID NO: 2 accordingto an embodiment of the present invention;

FIG. 7 is a graph that confirms a tyrosinase activity increasing effectof a peptide consisting of the amino acid sequence of SEQ ID NO: 2according to an embodiment of the present invention;

FIG. 8 is a graph that confirms a melanogenesis-related gene expressionincreasing effect of a peptide consisting of the amino acid sequence ofSEQ ID NO: 2 according to an embodiment of the present invention;

FIG. 9 shows a melanogenesis-related protein expression increasingeffect of a peptide consisting of the amino acid sequence of SEQ ID NO:2 according to an embodiment of the present invention;

FIG. 10 shows an effect of increased phosphorylation of CREB of apeptide consisting of the amino acid sequence of SEQ ID NO: 2 accordingto an embodiment of the present invention;

FIG. 11 is a graph that confirms a melanogenesis increasing effect of apeptide consisting of the amino acid sequence of SEQ ID NO: 3 accordingto an embodiment of the present invention;

FIG. 12 is a graph that confirms a tyrosinase activity increasing effectof a peptide consisting of the amino acid sequence of SEQ ID NO: 3according to an embodiment of the present invention;

FIG. 13 is a graph that confirms a melanogenesis-related gene expressionincreasing effect of a peptide consisting of the amino acid sequence ofSEQ ID NO: 3 according to an embodiment of the present invention;

FIG. 14 shows a melanogenesis-related protein expression increasingeffect of a peptide consisting of the amino acid sequence of SEQ ID NO:3 according to an embodiment of the present invention; and

FIG. 15 shows an effect of increased phosphorylation of CREB of apeptide consisting of the amino acid sequence of SEQ ID NO: 3 accordingto an embodiment of the present invention.

BEST MODE

Provided is a peptide consisting of the amino acid sequence of SEQ IDNO: 1, 2, or 3.

MODE OF DISCLOSURE

Hereinafter, the present invention will be described in detail withreference to examples. However, these examples are only for illustrativepurposes, and the scope of the present invention is not limited by theseexamples.

Synthetic Example 1: Peptide Synthesis

70 g of chlorotrityl chloride resin (CTC resin, Nova Biochem Cat No.01-64-0021) was added into a reaction container, and 490 ml of methylenechloride (MC) was added followed by stirring for 3 minutes. After thesolution was removed, 490 ml of dimethyl formamide (DMF) was added,followed by stirring for 3 minutes, and then the solvent was againremoved. 700 ml of a dichloromethane solution was added to a reactioncontainer, and 200 mmole of Fmoc-Leu-OH (Bachem, Swiss) and 400 mmole ofdiisopropyl ethylamine (DIEA) were added. Thereafter, the mixture waswell dissolved with stirring, and then the reaction was conducted withstirring for 1 hour. After the reaction, washing was conducted, and thenmethanol and DIEA (2:1) were dissolved in dichloromethane (DCM),followed by reaction for 10 minutes, and then the resultant was washedwith excess DCM/DMF (1:1). After the solution was removed, 490 ml ofdimethyl formamide (DMF) was added, followed by stirring for 3 minutes,and then the solvent was again removed. 700 ml of a deprotectionsolution (20% piperidine/DMF) was added to a reaction container,followed by stirring at room temperature for 10 minutes, and then thesolution was removed. An equal amount of a deprotection solution wasadded, and then the reaction was again maintained for 10 minutes, andthereafter, the solution was removed, followed by washing twice withDMF, once with MC, and once with DMF, for 3 minutes each, therebypreparing Leu-CTC resin.

700 ml of a DMF solution was added to a new reaction container, and 200mmol Fmoc-Ser(tBu)-OH (Bachem, Swiss), 200 mmol HoBt, and 200 mmole ofHBTu were added, and the mixture was well dissolved with stirring. 400mmole DIEA was added to the reaction container in two divided portions,and then stirring was conducted for at least 5 minutes until all solidswere dissolved. The dissolved amino acid mixed solution was added to thereaction container containing the deprotected resin, and the reactionwas conducted with stirring at room temperature for 1 hour. After thereaction solution was removed, the stirring was conducted using a DMFsolution three times for 5 minutes each, followed by removal. A smallamount of the reaction resin was taken to check the extent of reactionusing the Kaiser test (Ninhydrin test). The deprotection reaction wastwice conducted using a deprotection solution in the same manner asdescribed above, thereby preparing Ser(tBu)-Leu-CTC resin. Aftersufficient washing with DMF and MC, the Kaiser test was again conducted,and then the following amino acid attachment test was conducted in thesame manner as described above. A chain reaction was conducted in theorder of Fmoc-Trp-OH, Fmoc-Ile-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Pro-OH,Fmoc-Cys(Trt)-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Leu-OH,Fmoc-Cys(Trt)-OH, Fmoc-Phe-OH on the basis of the selected amino acidsequence. The Fmoc-protecting group was removed by reaction twice withthe deprotection solution for 10 minutes for each and then favorablewashing. The peptidyl resin was washed with DMF, MC, and methanol threetimes for each, dried under the flow of nitrogen gas, and completelydried by decompression under vacuum in P₂O₅. Thereafter, 1,900 ml of aleaving solution [81.5% of trifluoroacetic acid (TFA), 5.0% of distilledwater, 5.0% of thioanisole, 5.0% of phenol, 2.5% of ethanedithiol (EDT),and 1.0% of triisopropylsilane (TIS)] was added, and the reaction wasmaintained for 2 hours while stirring the mixture at room temperature.The resin was obtained through filtration, washed with a small amount ofTFA solution, and then mixed with the stock solution. Cold ether wasadded to 2,090 ml of the resultant with the stock solution to induceprecipitation, and the precipitates were collected by centrifugation,followed by washing twice with cold ether. The stock solution wasremoved, followed by sufficient drying under nitrogen atmosphere, andthus 129.8 g of a peptide consisting of the amino acid sequence of SEQID NO: 1 (yield: 92.8%) was synthesized, before purification. Themolecular weight was determined as 1398.6 (theoretical value: 1398.6) byusing a molecular weight analysis system.

In addition, the peptide composed of the amino acid sequence of SEQ IDNO: 2 or SEQ ID NO: 3 was synthesized in the same manner as describedabove.

TABLE 1  Analysis value SEQ (Mass spectrometer) ID Sequence listing Analytical Theoretical NO: (5′->3′) value value 1 FCLGPCPYIWSL 1398.61398.6 2 KVTAMRCFLL 1181.5 1181.5 3 RVTAMRCFLL 1209.5 1209.5

Example 1: Melanogenesis Assay

After seeding melanocytes (B16F10 cell line) in 6-well plates at thedensity of 5×10⁴ cells/well, the melanocytes were cultured in anincubator at a temperature of 37° C. for 24 hours, and the medium ofeach plate was removed and replaced with 2% serum-containing media,followed by treatment with the present peptide at differentconcentrations and then incubation of 72 hours.

Then, the culture medium was removed, and the cells were taken off andthen transferred into 1.5-ml tubes, followed by centrifugation at 13,000rpm for 3 minutes to remove the supernatant. Next, cell pellets werecollected to observe melanin. 150 μl of 1 N NaOH was added to the cellpellets to lyse intracellular melanin at a temperature of 60° C. for 30minutes. Thereafter, 100 μl of the supernatant obtained from the lysiswas added into each well of 96-well plates, and the absorbance wasmeasured at 450 nm, and the results are shown in FIGS. 1, 6, and 11 andTables 2 and 4.

TABLE 2 Control SEQ ID NO: 1 SEQ ID NO: 1 a-MSH group (10 μM) (50 μM)(200 nM) Melanin 100 125 264 276 content (%)

TABLE 3 Control SEQ ID NO: 2 SEQ ID NO: 2 a-MSH group (10 μM) (50 μM)(200 nM) Melanin 100 109 155 263 content (%)

TABLE 4 Control SEQ ID NO: 3 SEQ ID NO: 3 a-MSH group (10 μM) (50 μM)(200 nM) Melanin 100 106 164 263 content (%)

As it may be confirmed in FIGS. 1, 6, and 11 and Tables 2 and 4,melanogenesis was increased when mouse melanin cell line B16F10 wastreated with the peptide consisting of the amino acid sequence of SEQ IDNO: 1, 2, or 3.

Example 2: Tyrosinase Activity Assay

Melanoma cell line (B16F10) cells were cultured in 6-well culture platesfor 24 hours, and treated with the peptide with differentconcentrations, followed by culture for 72 hours. The 6-well cultureplates were loaded on ice and washed with cool PBS, and then 300 μl of0.1 M sodium phosphate buffer (pH 6.8, lysis buffer) containing 1%Triton X-100 was added. The cells were collected in 1.5-ml tubes, andthen cell membranes were disrupted by repeating five timesrapid-freezing at −270° C. and thawing. After centrifugation at 15,000rpm for 10 minutes, the supernatant was collected in other 1.5-mL tubes,and the protein of the samples was quantified. The samples were dilutedto have the same protein concentration and then dispensed in every threewells in 96-well culture plates, and then 20 μl of 10 mM L-DOPA wasadded, followed by incubation at 37° C. for 1 hour The blank andpositive control were as shown in Table 5.

TABLE 5 Sample Blank Positive control Sample 90 μl — — Buffer — 90 μl 80μl Mushroom tyrosinase — — 10 μl (0.1 mg/ml)

Then, the absorbance was measured at 475 nm, and the results are shownin FIGS. 2, 7, and 12 , and Tables 6 to 8.

TABLE 6 Control SEQ ID NO: 1 SEQ ID NO: 1 a-MSH group (10 μM) (50 μM)(200 nM) Tyrosinase 100 157 201 467 activity (%)

TABLE 7 Control SEQ ID NO: 2 SEQ ID NO: 2 a-MSH group (10 μM) (50 μM)(200 nM) Tyrosinase 100 144 189 484 activity (%)

TABLE 8 Control SEQ ID NO: 3 SEQ ID NO: 3 a-MSH group (10 μM) (50 μM)(200 nM) Tyrosinase 100 114 173 484 activity (%)

As it may be confirmed in FIGS. 2, 7, and 12 and Tables 6 to 8,tyrosinase activity was increased when mouse melanin cell line B16F10was treated with the peptide composed of the amino acid sequence of SEQID NO: 1, 2, or 3.

Example 3: RT-PCR of Melanogenesis-Related Genes

Melanocytes (B16F10 cell line) were seeded on 6-well culture plates atthe density of 5×10⁴ cells/well and incubated in an incubator for 24hours. Then, the medium was replaced with 00, and the cells were treatedwith the peptides of the present invention with different concentrationsand cultured for 72 hours. Next, after RNA extraction of cells andquantification, cDNA was synthesized by using the cDNA synthesis kit(Intron, Korea). Thereafter, as shown in Table 3, PCR was performedusing specific primers for MITF, tyrosinase, and TRP1, which are factorsinvolved in melanogenesis. Then, degrees of mRNA expression of thegrowth factors under each sample treatment conditions were compared byrunning the resultants on a 5% agarose gel, and the results are shown inFIGS. 3, 8, and 13 .

TABLE 9  SEQ ID NO: Primer name Sequence listing (5′-3′) 4 MITF_FCCAGCCTGGCGATCATGTCAT 5 MITF_R GGTCTGGACAGGAGTTGCTG 6 tyrosinase_FGGCCAGCTTTCAGGCAGAGG 7 tyrosinase_R TGGTGCTTCATGGGCAAAAT 8 TRP1_FTCTGTGAAGGTGTGCAGGAG 9 TRP1_R CCGAAACAGAGTGGAAGGTT

As it may be confirmed in FIGS. 3, 8, and 13 , the mRNA expression ofMITF, tyrosinase, and TRP1, which are transcriptional factors involvedin melanogenesis, were increased when the mouse melanin cell line B16F10was treated with the peptide composed of the amino acid sequence of SEQID NO: 1, 2, or 3.

Example 4: Western Blotting of Melanogenesis-Related Proteins

Melanocytes (B16F10 cell line) were seeded on 6-well culture plates atthe density of 5×10⁴ cells/well and incubated in an incubator for 24hours, and the cells were treated with the peptides of the presentinvention with different concentrations. After 72-hour incubation, thecells were lysed, and the cells were subjected to western blotting usingantibodies (antacruz biotechnology, USA) each specific to MITF andtyrosinase, which are the factors involved in melanogenesis. The resultsare shown in FIGS. 4, 9 , and 14.

As it may be confirmed in FIGS. 4, 9, and 14 , the protein expressioninvolved in the process of melanogenesis increased when the mousemelanin cell line B16F10 was treated with the peptide composed of theamino acid sequence of SEQ ID NO: 1, 2, or 3.

Example 5: Melanogenesis-Related Protein Activity Assay

Melanocytes (B16F10 cell line) were seeded on 6-well culture plates atthe density of 5×10⁴ cells/well and incubated in an incubator for 24hours, and the cells were treated with the peptides of the presentinvention with different concentrations. After 48-hour incubation, thecells were lysed, and the cells were subjected to western blotting usingspecific antibodies (Cell Signaling Technology, USA) to investigate thephosphorylation level of CREB, which is a signaling substance involvedin melanogenesis. The results are shown in FIGS. 5, 10, and 15 .

As it may be confirmed in FIGS. 5, 10, and 15 , the phosphorylationlevel of CREB, which is a factor involved in melanogenesis, wasincreased when the mouse melanin cell line B16F10 was treated with thepeptide composed of the amino acid sequence of SEQ ID NO: 1, 2, or 3.

INDUSTRIAL APPLICABILITY

The present invention is related to a peptide showing melanogenesispromoting activity; a pharmaceutical composition for prevention and/ortreatment of hypomelanosis including the peptide as an activeingredient; a cosmetic composition for prevention and/or alleviation ofhypomelanosis including the peptide as an active ingredient; and the useof the peptide for prevention, alleviation, and/or treatment ofhypomelanosis.

The invention claimed is:
 1. A peptide that has an activity againsthypomelanosis and consists of an amino acid sequence selected from thegroup consisting of SEQ ID NO: 2 and SEQ ID NO: 3, wherein optionallythe peptide includes a modification at the N-terminal and/or C-terminalend.
 2. The peptide of claim 1, where the peptide includes saidN-terminal and/or C-terminal modification.
 3. The peptide of claim 2,wherein the peptide includes an N-terminal modification, which is aprotecting group.
 4. The peptide of claim 3, wherein the protectinggroup is selected from the group consisting of an acetyl group, afluorenyl methoxy carbonyl group, a formyl group, a palmitoyl group, amyristyl group, a stearyl group, and polyethylene glycol (PEG).
 5. Thepeptide of claim 2, wherein the peptide includes a C-terminalmodification selected from the group consisting of an amino group (—NH₂)and an azide group (—NHNH₂).
 6. A pharmaceutical composition for thetreatment of hypomelanosis, comprising, as an active ingredient, atleast one peptide consisting of a sequence selected from the groupconsisting of SEQ ID NO: 2 and SEQ ID NO: 3, wherein optionally thepeptide includes a modification at the N-terminal and/or C-terminal end.7. The pharmaceutical composition of claim 6, wherein the hypomelanosisis selected from the group consisting of vitiligo, albinism, nevusdepigmentosus, pityriasis alba, pityriasis versicolor, post-inflammatorydepigmentation, morphea, piebaldism, idiopathic guttate hypomelanosis,and leucoderma punctatum.
 8. The pharmaceutical composition of claim 6,where the peptide includes said N-terminal and/or C-terminalmodification.
 9. The pharmaceutical composition of claim 8, wherein thepeptide includes an N-terminal modification, which is a protectinggroup.
 10. The pharmaceutical composition of claim 9, wherein theprotecting group is selected from the group consisting of an acetylgroup, a fluorenyl methoxy carbonyl group, a formyl group, a palmitoylgroup, a myristyl group, a stearyl group, and polyethylene glycol (PEG).11. The pharmaceutical composition of claim 8, wherein the peptideincludes a C-terminal modification selected from the group consisting ofan amino group (—NH₂) and an azide group (—NHNH₂).
 12. A cosmeticcomposition for the alleviation of hypomelanosis, comprising, as anactive ingredient, at least one peptide consisting of a sequenceselected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 3,wherein optionally the peptide includes a modification at the N-terminaland/or C-terminal end.
 13. The cosmetic composition of claim 12, whereinthe hypomelanosis is selected from the group consisting of vitiligo,albinism, nevus depigmentosus, pityriasis alba, pityriasis versicolor,post-inflammatory depigmentation, morphea, piebaldism, idiopathicguttate hypomelanosis, and leucoderma punctatum.
 14. The cosmeticcomposition of claim 12, where the peptide includes said N-terminaland/or C-terminal modification.
 15. The cosmetic composition of claim14, wherein the peptide includes an N-terminal modification, which is aprotecting group.
 16. The cosmetic composition of claim 15, wherein theprotecting group is selected from the group consisting of an acetylgroup, a fluorenyl methoxy carbonyl group, a formyl group, a palmitoylgroup, a myristyl group, a stearyl group, and polyethylene glycol (PEG).17. The cosmetic composition of claim 14, wherein the peptide includes aC-terminal modification selected from the group consisting of an aminogroup (—NH₂) and an azide group (—NHNH₂).
 18. A method for the treatmentor alleviation of hypomelanosis in a subject in need thereof, the methodcomprising administering the peptide of claim 1 to the subject.
 19. Themethod of claim 18, wherein the peptide is comprised within apharmaceutical composition or a cosmetic composition.
 20. The method ofclaim 18, wherein the hypomelanosis is selected from the groupconsisting of vitiligo, albinism, nevus depigmentosus, pityriasis alba,pityriasis versicolor, post-inflammatory depigmentation, morphea,piebaldism, idiopathic guttate hypomelanosis, and leucoderma punctatum.21. The method of claim 18, where the peptide includes said N-terminaland/or C-terminal modification.
 22. The method of claim 21, wherein thepeptide includes an N-terminal modification, which is a protectinggroup.
 23. The method of claim 22, wherein the protecting group isselected from the group consisting of an acetyl group, a fluorenylmethoxy carbonyl group, a formyl group, a palmitoyl group, a myristylgroup, a stearyl group, and polyethylene glycol (PEG).
 24. The method ofclaim 21, wherein the peptide includes a C-terminal modificationselected from the group consisting of an amino group (—NH₂) and an azidegroup (—NHNH₂).